Successful production of proteins of interest (POI) has been accomplished both with prokaryotic and eukaryotic hosts. The most prominent examples are bacteria like Escherichia coli, yeasts like Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi like Aspergillus awamori or Trichoderma reesei, or mammalian cells like CHO cells. While the yield of some proteins is readily achieved at high rates, many other proteins are only produced at comparatively low levels.
To improve the secretion of a recombinant protein, one strategy is to target on the host's secretory pathway involving the folding and processing of proteins.
Co-expression of a cDNA encoding protein disulfide isomerase (PDI) and a cDNA encoding a heterologous disulphide-bonded protein was first suggested in WO 93/25676 as a means to increase the yield of the heterologous protein. WO 93/25676 reported that the recombinant expression of antistasin and tick anticoagulant protein can be increased by co-expression with PDI.
WO 94/08012 provided methods for increasing the secretion of overexpressed protein in yeast by increasing expression of an Hsp70 chaperone protein, i.e. KAR2/BiP, or a PDI chaperone protein.
WO 05/0617818 and WO 06/067511 provided methods for producing a desired heterologous protein in yeast by using a 2 μm-based expression plasmid. It was demonstrated that the yield of a heterologous protein is substantially increased when the genes for one or more chaperone protein(s) and a heterologous protein are co-expressed on the same plasmid.
WO 2008/128701A2 described an expression system to increase the secretion of a POI from a eukaryotic cell by employing one of the following proteins BMH2, BFR2, COG6, C0Y1, CUP5, IMH 1, KIN2, SEC31, SSA4 and SSE1.
Another approach to increase protein production is based on the overexpression of HAC1, a transcription factor that activates the unfolded protein response (UPR). Transcriptional analyses revealed that more than 330 genes are regulated by HAC1, most of them being involved in secretion or in the biogenesis of secretory organelles. WO 01/72783 describes methods for increasing the amount of a heterologous protein secreted from a eukaryotic cell by inducing an elevated unfolded protein response (UPR), wherein the UPR is modulated by co-expression of a protein selected from the group consisting of HAC1, PTC2 and IRE1.
Wentz et al. employed a Saccharomyces cerevisiae yeast surface display gene library to identify improved secretion strains by applying an appropriate selection pressure. The yeast cDNAs CCW12, SED1, CWP2, RPP0 were found to enhance the display of scTCR in a temperature-dependent manner. ERO1 enhanced protein secretion when induced at 20° C. (Wentz et al., Appl. Environ. Microbiol. (2007) 73(4):1189-1198).
Liu et al. (Biotechnol. Prog. (2006), 22:1090-1095) showed that co-overexpression of Kar2 in Pichia pastoris increases rhG-CSF expression 5.6 fold. Combining KAR2 with Sec63, PDI and YDJ resulted in an increase of 2.8, 6.5 and 5.94 fold. In the experiments performed by Blatt et al, co-overexpression of KAR2 (BiP) increased A33scFv expression in Pichia pastoris two-fold. Combining KAR2 with PDI almost eliminated the positive KAR2 effect (Blatt et al., Appl. Microbiol. Biotechnol., 2007, 74:381-389).
Guerfall et al. examined the effect of overexpressing endogenous HAC1 in P. pastoris. Furthermore, HAC1 was overexpressed constitutively and inducibly. In all cases, an increased KAR2 expression as a result of induced UPR was identified. Constitutive overexpression of full-length HAC1 had little or no effect, while overexpression of the induced form of HAC1 led to an increase of protein secretion in one out of four cases, and a decrease in three out of four cases (Guerfall et al., Microbial Cell Factories, (2010), 9:49).
Sleep et al. showed that co-overexpression of Lhs1 increases the concentration of rHA. LHS1 was combined with SIL1, JEM1 and SCJ1, but titers were lower than with JEM1 co-overexpressed alone. Co-overexpression of SIL1, LHS1 and JEM1 at the same time increased GM-CSF expression by 1.45 fold and the rHA expression by approximately 1.1 and 2 fold, dependent on the cultivation media (Sleep et al. Applied and Environmental Microbiology, (2008) 74(24):7759-7766).
US 2009221030 A1 described the co-expression of various helper proteins, inter alia, BiP1, alone and in combination with other helper proteins, inter alia, LHS1, in Trichoderma reesei. The highest expression values were obtained with BiP1 alone, while the combination of BiP1 and LHS1 lead to 8% lower secreted protein titers.
U.S. Pat. No. 8,440,456 provided the genome sequence of Pichia pastoris and disclosed nucleic acid sequences encoding signal peptides, chaperons and promoters. It disclosed expression vectors comprising the nucleic acid sequences and genetically engineered yeast capable of overexpression of 14 chaperones. ROT1, SHR3 and SIL1 were specifically selected for testing, however, no expression was observed for SIL1, and overexpression of ROT1 or SHR3 failed to lead to any significant enhancement of the secretion of heterologous proteins. While overexpression of genes encoding helper proteins is usually applied to enhance expression of a protein of interest, underexpression of a gene encoding a protein or even a knock-out of the gene encoding it is not or only rarely applied.
High level of protein yield in host cells may be limited at one or more different steps, like folding, disulfide bond formation, glycosylation, transport within the cell, or release from the cell. Many of the mechanisms involved are still not fully understood and cannot be predicted on the basis of the current knowledge of the state-of-the-art, even when the DNA sequence of the entire genome of a host organism is available.
There is a constant need for methods to improve a host cell's capacity to produce and/or secret proteins of interest. One object of the invention is to provide new methods to increase the yield of proteins in host cells which are simple and efficient and suitable for use in industrial methods. It is another object to provide host cells to achieve this purpose.
It must be noted that as used herein, the singular forms “a”, “an” and “the” include plural references and vice versa unless the context clearly indicates otherwise. Thus, for example, a reference to “a host cell” or “a method” includes one or more of such host cells or methods, respectively, and a reference to “the method” includes equivalent steps and methods that could be modified or substituted known to those of ordinary skill in the art. Similarly, for example, a reference to “methods” or “host cells” includes “a host cell” or “a method”, respectively.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”. For example, A, B and/or C means A, B, C, A+B, A+C, B+C and A+B+C.
The term “about” or “approximately” as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes also the concrete number, e.g., about 20 includes 20.
The term “less than”, “more than” or “larger than” includes the concrete number. For example, less than 20 means 20 and more than 20 means 20.
Throughout this specification and the claims or items, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer (or step) or group of integers (or steps). It does not exclude any other integer (or step) or group of integers (or steps). When used herein, the term “comprising” can be substituted with “containing”, “composed of”, “including”, “having” or “carrying.” When used herein, “consisting of” excludes any integer or step not specified in the claim/item. When used herein, “consisting essentially of” does not exclude integers or steps that do not materially affect the basic and novel characteristics of the claim/item. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms.
Further, in describing representative embodiments of the present invention, the specification may have presented the method and/or process of the present invention as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process of the present invention should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the present invention.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein. The terminologies used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined solely by the claims/items.
All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.